The proposed project should enhance candidate's previous experience in vascular biology and diabetes and develop the candidate into an independent researcher. While Pl's experience with in vivo and in vitro experimental techniques will help to conduct the presented project, its completion will significantly enrich candidate's knowledge in cell biology and physiology as well. Endothelial cell dysfunction (ECD) is a common precursor and denominator of various pathologic conditions, including stroke, hypertension, myocardial infarction and diabetes mellitus. The established hallmarks of endothelial cell dysfunction are a deficiency in the production or bioavailability of nitric oxide (NO), an increased level of plasminogen activator inhibitor-1 (PAl-1), and a presence of a pro-coagulant state. We have previously demonstrated that elevated level of PAl-1 results in a decreased production of NO by cultured endothelial cells, as well as an increased production of microparticies stained with Annexin V - evidence for the expression of anionic phospholipids. This was accompanied by the accelerated generation of thrombin by microparticles. PAl-1 knockout mice presented with a significantly decreased number of circulating microparticles and a marked increase in the number of microparticles after PAl-1 injection. Combined with recent findings of elevated levels of circulating endothelial microparticles in patients with acute coronary syndrome and diabetes, our data suggest that a) microparticles may be a consequence of ECD and contribute to a procoagulant state accompanying these diseases and b) raise the question whether increased levels of circulating microparticles may contribute to the development and maintenance of endothelial cell dysfunction. Based on these findings we hypothesize that: 1) increased microparticle formation by stressed endothelial cells is not only a novel marker of, but also a contributor to endothelial dysfunction and 2) elevated level of PAl-1 (a recognized marker of endothelial dysfunction) leads to increased formation of microparticles with pro-coagulant properties. The current proposal seeks to 1) establish a novel pathophysiolgical mechanism of endothelial dysfunction - formation of microparticles by stressed endothelial cells; 2) obtain information on the population of circulating microparticles produced by endothelial cells in diabetes mellitus; 3) to investigate possible links between identified markers of endothelial dysfunction, namely elevated levels of PAl-1 and increased coagulant activity. To accomplish these goals, microparticle formation will be studied using fluorescence-activated cell sorting in vitro (in endothelial cells stressed with high glucose or PAl-1) and in vivo (in Zucker diabetic rats). The link between PAl-1 and increased coagulant activity will be studied in vivo using PAl-1 knockout mice and in vitro using endothelial cell cultures. The proposed studies may provide novel information on the contribution of microparticles to the pathophysiology of endothelial cell dysfunction and coagulation abnormalities.